Hydroxysteroid 17-ß dehydrogenase 14 (HSD17B14) is an L-fucose dehydrogenase, the initial enzyme of the L-fucose degradation pathway.

L-Fucose (6-deoxy-L-galactose), a monosaccharide abundant in glycolipids and glycoproteins produced by mammalian cells, has been extensively studied for its role in intracellular biosynthesis and recycling of GDP-L-fucose for fucosylation. However, in certain mammalian species, L-fucose is efficiently broken down to pyruvate and lactate in a poorly understood metabolic pathway. In the 1970s, L-fucose dehydrogenase, an enzyme responsible for the initial step of this pathway, was partially purified from pig and rabbit livers and characterized biochemically. However, its molecular identity remained elusive until recently. This study reports the purification, identification, and biochemical characterization of the mammalian L-fucose dehydrogenase. The enzyme was purified from rabbit liver approximately 340-fold. Mass spectrometry analysis of the purified protein preparation identified mammalian hydroxysteroid 17-ß dehydrogenase 14 (HSD17B14) as the sole candidate enzyme. Rabbit and human HSD17B14 were expressed in HEK293T and Escherichia coli, respectively, purified and demonstrated to catalyze the oxidation of L-fucose to L-fucono-1,5-lactone, as confirmed by mass spectrometry and NMR analysis. Substrate specificity studies revealed that L-fucose is the preferred substrate for both enzymes. The human enzyme exhibited a catalytic efficiency for L-fucose that was 359-fold higher than its efficiency for estradiol. Additionally, recombinant rat HSD17B14 exhibited negligible activity towards L-fucose, consistent with the absence of L-fucose metabolism in this species. The identification of the gene encoding mammalian L-fucose dehydrogenase provides novel insights into the substrate specificity of enzymes belonging to the 17-ß-hydroxysteroid dehydrogenase family. This discovery also paves the way for unraveling the physiological functions of the L-fucose degradation pathway, which remains enigmatic.

Nucleolar Essential Protein 1 (Nep1): Elucidation of enzymatic catalysis mechanism by molecular dynamics simulation and quantum mechanics study.

The Nep1 protein is essential for the formation of eukaryotic and archaeal small ribosomal subunits, and it catalyzes the site-directed SAM-dependent methylation of pseudouridine (Ψ) during pre-rRNA processing. It possesses a non-trivial topology, namely, a 31 knot in the active site. Here, we address the issue of seemingly unfeasible deprotonation of Ψ in Nep1 active site by a distant aspartate residue (D101 in S. cerevisiae), using a combination of bioinformatics, computational, and experimental methods. We identified a conserved hydroxyl-containing amino acid (S233 in S. cerevisiae, T198 in A. fulgidus) that may act as a proton-transfer mediator. Molecular dynamics simulations, based on the crystal structure of S. cerevisiae, and on a complex generated by molecular docking in A. fulgidus, confirmed that this amino acid can shuttle protons, however, a water molecule in the active site may also serve this role. Quantum-chemical calculations based on density functional theory and the cluster approach showed that the water-mediated pathway is the most favorable for catalysis. Experimental kinetic and mutational studies reinforce the requirement for the aspartate D101, but not S233. These findings provide insight into the catalytic mechanisms underlying proton transfer over extended distances and comprehensively elucidate the mode of action of Nep1.

Are there double knots in proteins? Prediction and in vitro verification based on TrmD-Tm1570 fusion from C. nitroreducens.

We have been aware of the existence of knotted proteins for over 30 years-but it is hard to predict what is the most complicated knot that can be formed in proteins. Here, we show new and the most complex knotted topologies recorded to date-double trefoil knots (31 #31). We found five domain arrangements (architectures) that result in a doubly knotted structure in almost a thousand proteins. The double knot topology is found in knotted membrane proteins from the CaCA family, that function as ion transporters, in the group of carbonic anhydrases that catalyze the hydration of carbon dioxide, and in the proteins from the SPOUT superfamily that gathers 31 knotted methyltransferases with the active site-forming knot. For each family, we predict the presence of a double knot using AlphaFold and RoseTTaFold structure prediction. In the case of the TrmD-Tm1570 protein, which is a member of SPOUT superfamily, we show that it folds in vitro and is biologically active. Our results show that this protein forms a homodimeric structure and retains the ability to modify tRNA, which is the function of the single-domain TrmD protein. However, how the protein folds and is degraded remains unknown.

NMR Assignment of Methyl Groups in Immobilized Proteins Using Multiple-Bond 13C Homonuclear Transfers, Proton Detection, and Very Fast MAS.

In nuclear magnetic resonance spectroscopy of proteins, methyl protons play a particular role as extremely sensitive reporters on dynamics, allosteric effects, and protein-protein interactions, accessible even in high-molecular-weight systems approaching 1 MDa. The notorious issue of their chemical shift assignment is addressed here by a joint use of solid-state 1H-detected methods at very fast (nearly 100 kHz) magic-angle spinning, partial deuteration, and high-magnetic fields. The suitability of a series of RF schemes is evaluated for the efficient coherence transfer across entire 13C side chains of methyl-containing residues, which is key for establishing connection between methyl and backbone 1H resonances. The performance of ten methods for recoupling of either isotropic 13C-13C scalar or anisotropic dipolar interactions (five variants of TOBSY, FLOPSY, DIPSI, WALTZ, RFDR, and DREAM) is evaluated experimentally at two state-of-the-art magic-angle spinning (55 and 94.5 kHz) and static magnetic field conditions (18.8 and 23.5 T). Model isotopically labeled compounds (alanine and Met-Leu-Phe tripeptide) and ILV-methyl and amide-selectively protonated, and otherwise deuterated chicken α-spectrin SH3 protein are used as convenient reference systems. Spin dynamics simulations in SIMPSON are performed to determine optimal parameters of these RF schemes, up to recently experimentally attained spinning frequencies (200 kHz) and B 0 field strengths (28.2 T). The concept of linearization of 13C side chain by appropriate isotope labeling is revisited and showed to significantly increase sensitivity of methyl-to-backbone correlations. A resolution enhancement provided by 4D spectroscopy with non-uniform (sparse) sampling is demonstrated to remove ambiguities in simultaneous resonance assignment of methyl proton and carbon chemical shifts.

Cotranslocational processing of the protein substrate calmodulin by an AAA+ unfoldase occurs via unfolding and refolding intermediates.

Protein remodeling by AAA+ enzymes is central for maintaining proteostasis in a living cell. However, a detailed structural description of how this is accomplished at the level of the substrate molecules that are acted upon is lacking. Here, we combine chemical cross-linking and methyl transverse relaxation-optimized NMR spectroscopy to study, at atomic resolution, the stepwise unfolding and subsequent refolding of the two-domain substrate calmodulin by the VAT AAA+ unfoldase from Thermoplasma acidophilum By engineering intermolecular disulphide bridges between the substrate and VAT we trap the substrate at different stages of translocation, allowing structural studies throughout the translocation process. Our results show that VAT initiates substrate translocation by pulling on intrinsically unstructured N or C termini of substrate molecules without showing specificity for a particular amino acid sequence. Although the B1 domain of protein G is shown to unfold cooperatively, translocation of calmodulin leads to the formation of intermediates, and these differ on an individual domain level in a manner that depends on whether pulling is from the N or C terminus. The approach presented generates an atomic resolution picture of substrate unfolding and subsequent refolding by unfoldases that can be quite different from results obtained via in vitro denaturation experiments.

Structure of a AAA+ unfoldase in the process of unfolding substrate.

AAA+ unfoldases are thought to unfold substrate through the central pore of their hexameric structures, but how this process occurs is not known. VAT, the Thermoplasma acidophilum homologue of eukaryotic CDC48/p97, works in conjunction with the proteasome to degrade misfolded or damaged proteins. We show that in the presence of ATP, VAT with its regulatory N-terminal domains removed unfolds other VAT complexes as substrate. We captured images of this transient process by electron cryomicroscopy (cryo-EM) to reveal the structure of the substrate-bound intermediate. Substrate binding breaks the six-fold symmetry of the complex, allowing five of the six VAT subunits to constrict into a tight helix that grips an ~80 Å stretch of unfolded protein. The structure suggests a processive hand-over-hand unfolding mechanism, where each VAT subunit releases the substrate in turn before re-engaging further along the target protein, thereby unfolding it.

Unfolding the mechanism of the AAA+ unfoldase VAT by a combined cryo-EM, solution NMR study.

The AAA+ (ATPases associated with a variety of cellular activities) enzymes play critical roles in a variety of homeostatic processes in all kingdoms of life. Valosin-containing protein-like ATPase of Thermoplasma acidophilum (VAT), the archaeal homolog of the ubiquitous AAA+ protein Cdc48/p97, functions in concert with the 20S proteasome by unfolding substrates and passing them on for degradation. Here, we present electron cryomicroscopy (cryo-EM) maps showing that VAT undergoes large conformational rearrangements during its ATP hydrolysis cycle that differ dramatically from the conformational states observed for Cdc48/p97. We validate key features of the model with biochemical and solution methyl-transverse relaxation optimized spectroscopY (TROSY) NMR experiments and suggest a mechanism for coupling the energy of nucleotide hydrolysis to substrate unfolding. These findings illustrate the unique complementarity between cryo-EM and solution NMR for studies of molecular machines, showing that the structural properties of VAT, as well as the population distributions of conformers, are similar in the frozen specimens used for cryo-EM and in the solution phase where NMR spectra are recorded.

Nuclear overhauser spectroscopy of chiral CHD methylene groups.

Nuclear magnetic resonance spectroscopy (NMR) can provide a great deal of information about structure and dynamics of biomolecules. The quality of an NMR structure strongly depends on the number of experimental observables and on their accurate conversion into geometric restraints. When distance restraints are derived from nuclear Overhauser effect spectroscopy (NOESY), stereo-specific assignments of prochiral atoms can contribute significantly to the accuracy of NMR structures of proteins and nucleic acids. Here we introduce a series of NOESY-based pulse sequences that can assist in the assignment of chiral CHD methylene protons in random fractionally deuterated proteins. Partial deuteration suppresses spin-diffusion between the two protons of CH2 groups that normally impedes the distinction of cross-relaxation networks for these two protons in NOESY spectra. Three and four-dimensional spectra allow one to distinguish cross-relaxation pathways involving either of the two methylene protons so that one can obtain stereospecific assignments. In addition, the analysis provides a large number of stereospecific distance restraints. Non-uniform sampling was used to ensure optimal signal resolution in 4D spectra and reduce ambiguities of the assignments. Automatic assignment procedures were modified for efficient and accurate stereospecific assignments during automated structure calculations based on 3D spectra. The protocol was applied to calcium-loaded calbindin D9k. A large number of stereospecific assignments lead to a significant improvement of the accuracy of the structure.

Distribution of Pico- and Nanosecond Motions in Disordered Proteins from Nuclear Spin Relaxation.

Intrinsically disordered proteins and intrinsically disordered regions (IDRs) are ubiquitous in the eukaryotic proteome. The description and understanding of their conformational properties require the development of new experimental, computational, and theoretical approaches. Here, we use nuclear spin relaxation to investigate the distribution of timescales of motions in an IDR from picoseconds to nanoseconds. Nitrogen-15 relaxation rates have been measured at five magnetic fields, ranging from 9.4 to 23.5 T (400-1000 MHz for protons). This exceptional wealth of data allowed us to map the spectral density function for the motions of backbone NH pairs in the partially disordered transcription factor Engrailed at 11 different frequencies. We introduce an approach called interpretation of motions by a projection onto an array of correlation times (IMPACT), which focuses on an array of six correlation times with intervals that are equidistant on a logarithmic scale between 21 ps and 21 ns. The distribution of motions in Engrailed varies smoothly along the protein sequence and is multimodal for most residues, with a prevalence of motions around 1 ns in the IDR. We show that IMPACT often provides better quantitative agreement with experimental data than conventional model-free or extended model-free analyses with two or three correlation times. We introduce a graphical representation that offers a convenient platform for a qualitative discussion of dynamics. Even when relaxation data are only acquired at three magnetic fields that are readily accessible, the IMPACT analysis gives a satisfactory characterization of spectral density functions, thus opening the way to a broad use of this approach.

Efficient determination of diffusion coefficients by monitoring transport during recovery delays in NMR.

A novel NMR approach allows one to efficiently determine translational diffusion coefficients of macromolecules in solution. This method for Signal Optimization with Recovery in Diffusion Delays (SORDID) monitors transport occurring during the recovery times between consecutive scans so that the duration of the measurements can be reduced approximately by a factor two.

Suppression of sampling artefacts in high-resolution four-dimensional NMR spectra using signal separation algorithm.

The development of non-uniform sampling (NUS) strategies permits to obtain high-dimensional spectra with increased resolution in significantly reduced experimental time. We extended a previously proposed signal separation algorithm (SSA) to process sparse four-dimensional NMR data. It is employed for two experiments carried out for a partially unstructured 114-residue construct of chicken Engrailed 2 protein, namely 4D HCCH-TOCSY and 4D C,N-edited NOESY. The SSA allowed us to obtain high-quality spectra using only as little as 0.16% of the available samples, with low sampling artefacts approaching the thermal noise level in most spectral regions. It is demonstrated that NUS 4D HCCH-TOCSY is dominated by sampling noise and requires efficient artefact suppression. On the other hand, 4D C,N-edited NOESY is a particularly attractive experiment for NUS, as the absence of diagonal peaks renders the problem of artefacts less critical. We also present a transverse-relaxation optimized sequence for HMQC that is especially designed for longer evolution periods in the indirectly detected proton dimension in high-dimensional pulse sequences. In conjunction with novel sampling strategies and efficient processing methods, this improvement enabled us to obtain unique structural information about aliphatic-amide contacts.

Methods to determine slow diffusion coefficients of biomolecules: applications to Engrailed 2, a partially disordered protein.

We present new NMR methods to measure slow translational diffusion coefficients of biomolecules. Like the heteronuclear stimulated echo experiment (XSTE), these new methods rely on the storage of information about spatial localization during the diffusion delay as longitudinal polarization of nuclei with long T(1) such as nitrogen-15. The new BEST-XSTE sequence combines features of Band-selective Excitation Short-Transient (BEST) and XSTE methods. By avoiding the saturation of all protons except those of amide groups, one can increase the sensitivity by 45% in small proteins. The new experiment which combines band-Selective Optimized Flip-Angle Short-Transient with XSTE (SOFAST-XSTE) offers an alternative when very short recovery delays are desired. A modification of the HSQC-edited version of the XSTE experiment offers enhanced sensitivity and access to higher resolution in the indirect dimension. These new methods have been applied to detect changes in diffusion coefficients due to dimerization or proteolysis of Engrailed 2, a partially disordered protein.

1H, 13C and 15N resonance assignment of a 114-residue fragment of Engrailed 2 homeoprotein, a partially disordered protein.

Engrailed 2 is a transcription factor belonging to the class of homeoproteins. These proteins possess a 60-residue DNA binding globular domain and play an important role in the early stages of development. We expressed and purified a 13.4 kDa fragment of Engrailed 2, which comprises a 54-residue N-terminal extension in addition to the homeodomain region. Almost all backbone and side-chain resonances have been assigned. The weak dispersion in the proton dimension of the (1)H-(15)N HSQC spectrum indicates the presence of disordered regions that do not belong to the homeodomain. This work is a first step toward the NMR investigation of the structure and dynamics of Engrailed 2 protein that contains a well-structured globular domain and partially disordered regions.

Can we trust docking results? Evaluation of seven commonly used programs on PDBbind database.

Docking is one of the most commonly used techniques in drug design. It is used for both identifying correct poses of a ligand in the binding site of a protein as well as for the estimation of the strength of protein-ligand interaction. Because millions of compounds must be screened, before a suitable target for biological testing can be identified, all calculations should be done in a reasonable time frame. Thus, all programs currently in use exploit empirically based algorithms, avoiding systematic search of the conformational space. Similarly, the scoring is done using simple equations, which makes it possible to speed up the entire process. Therefore, docking results have to be verified by subsequent in vitro studies. The purpose of our work was to evaluate seven popular docking programs (Surflex, LigandFit, Glide, GOLD, FlexX, eHiTS, and AutoDock) on the extensive dataset composed of 1300 protein-ligands complexes from PDBbind 2007 database, where experimentally measured binding affinity values were also available. We compared independently the ability of proper posing [according to Root mean square deviation (or Root mean square distance) of predicted conformations versus the corresponding native one] and scoring (by calculating the correlation between docking score and ligand binding strength). To our knowledge, it is the first large-scale docking evaluation that covers both aspects of docking programs, that is, predicting ligand conformation and calculating the strength of its binding. More than 1000 protein-ligand pairs cover a wide range of different protein families and inhibitor classes. Our results clearly showed that the ligand binding conformation could be identified in most cases by using the existing software, yet we still observed the lack of universal scoring function for all types of molecules and protein families.

An alternative flexible conformation of the E. coli HUß2 protein: structural, dynamics, and functional aspects.

The histone-like HU protein is the major nucleoid-associated protein involved in the dynamics and structure of the bacterial chromosome. Under physiological conditions, the three possible dimeric forms of the E. coli HU protein (EcHUα2, EcHUß2, and EcHUαß) are in thermal equilibrium between two dimeric conformations (N2 ↔ I2) varying in their secondary structure content. High-temperature molecular dynamics simulations combined with NMR experiments provide information about structural and dynamics features at the atomic level for the N2 to I2 thermal transition of the EcHUß2 homodimer. On the basis of these data, a realistic 3D model is proposed for the major I2 conformation of EcHUß2. This model is in agreement with previous experimental data.